The present invention relates to methods for identifying fungicides and also inhibitors of the 20S and 26S proteasomes, to methods for isolating 20S proteasomes, to the use of 20S proteasomes for identifying fungicides and to the use of the inhibitors of 26S and/or 20S proteasomes as fungicides. Although abbreviated citations are used hereinafter for convenience, the full citation of any abbreviated citation is available in the References section of this specification.
Undesired fungal growth which leads to considerable damage in agriculture every year, for example, can be controlled by the use of fungicides. The demands made on fungicides have increased constantly with regard to their activity, their costs and above all their ecological soundness. There exists therefore a demand for novel substances or classes of substances which can be developed into potent and ecologically sound novel fungicides. In general, it is customary to search for such novel lead structures in greenhouse tests. However, such tests are labour-intensive and expensive. The number of substances which can be tested in a greenhouse is, accordingly, limited. An alternative to such tests is the use of “high-throughput screening” (HTS) methods. This involves testing a large number of individual substances with regard to their effect on cells, individual gene products or genes in an automated method. When certain substances are found to have an effect, they can be studied in conventional screening methods and, where appropriate, developed further.
Ideal fungicides are, for example, those substances which inhibit gene products which have a decisive importance in the manifestation of the pathogenicity of a fungus. An example of such a fungicide is the active compound carpropamid which inhibits fungal melanin biosynthesis and thus prevents the formation of intact appressoria (adhesion organs). However, there is only a very small number of known gene products playing such a part in fungi. In addition, fungicides are known which lead to auxotrophism of the target cells by inhibiting corresponding biosynthetic pathways and, consequently, to the loss of pathogenicity.
Other important starting points are polypeptide or polypeptide complexes which play a central part in essential cellular processes or essential protein activities. The emphasis here is on the inhibition of enzymic reactions but it is also possible to inhibit the interaction of proteins or the functional interaction of complex protein machinery.
An example of such a complex “protein machinery” which is at the centre of cellular processes is the 26S proteasome. The biological function of the 26S proteasome comprises a multiplicity of functions, inter alia degradation of misfolded and misassembled proteins and peptides, regulation of stress response by, for example, degradation of transcription factors, or cell cycle control by the degradation of cyclins. For the cellular immune response, peptides are processed in such a way that they can be presented subsequently on the outer surface by the MHC I complex. The catalytic core of the 26S proteasome is the 20S proteasome. The 20S proteasome itself has various peptidase activities on which the enzymic activity of the 20S proteasome is based, as a result of which the 20S proteasome, in particular is an interesting target for substances which influence the said enzymic activities.
The proteolytic activity of the proteasome in vivo requires ubiquitination of the target proteins and consumes ATP. In this connection, the target proteins are degraded by transferring ubiquitin onto the target protein. The ubiquinated protein is then transported to the proteasome, where it is degraded and the ubiquitin is recycled.
It is known that all proteins of the 20S proteasome, except one in S. cerevisiae, are necessary for cell growth. The complex comprises a plurality of different proteolytic activities such as, for example, a peptidyl-glutamyl peptide-hydrolysing (PGPH) activity, a tryptic and a chymotryptic activity. Mutational analyses have revealed that the chymotryptic activity is the most important one in the complex. Previously, it has not been possible to express the 20S proteasome heterologously, but native purification of active 20S proteasomes from yeast have already been described.
The term “enzymic activity” of the 20S or 26S proteasome, as used herein, refers to at least one of the various enzymic activities of the 20S or 26S proteasome. Accordingly, an “enzymically active” 20S or 26S proteasome is still capable of carrying out at least one of the naturally occurring enzymic reactions.
The eukaryotic 26S proteasome consists of in each case one proteolytic and two regulatory complexes. The 20S proteasome is composed of 7 different α- and seven different β-subunits which have already been cloned from various organisms and sequenced. The 20S proteasome from baker's yeast (S. cerevisiae) has a molecular weight of approx. 700 kD. Likewise, various publications have already described the purification of proteasomes from eukaryotes (e.g. WO 98/42829 A1; EP 0 345 750 A2). The efforts to purify proteasomes in the best possible way finally also resulted in resolving of the structure of the 20S proteasome from S. cerevisiae (WO 98/42829 A1; Groll et al. (1997), Structure of the 20S proteasome from yeast at 2.4 Å resolution, Nature 386, 463-471).
Coux et al. (1998), (Enzymes catalyzing ubiquitination and proteolytic processing of the p105 precursor of nuclear factor kappaB1. J. Biol. Chem. 273(15), 8820-8828) describe the multiple proteolytic activities of the 20S proteasome as chymotrypsin-like, trypsin-like, PGPH, and as preferring branched amino acid chains and small neutral amino acids. The proteolytic activity can be enhanced by various induction conditions. These include, for example, heating the proteasome to 55° C. or the addition of SDS. Moreover, McCormack et al. (1998), Biochemistry 37, 7792-7800 describe various substrates for detecting the proteolytic activity of the 20S proteasome, such as, for example, Suc-Leu-Leu-Val-Tyr-AMC (SEQ. ID. NO. 1), Z-Leu-Leu-Arg-AMC and Z-Leu-Leu-Glu-2NA, where Suc is N-succinyl, AMC is 7-amino-methylcoumarin, Z is carbobenzyloxy and 2NA is 2-naphthylamine.
Publicly accessible databases such as MIPS (“Munich information centre for protein sequences”) or SGD (“saccharomyces genome database”) describe all subunits of the 26S proteasome, with a few exceptions, as essential.
Various reversible and irreversible inhibitors for the enzymic activity of proteasomes in vitro are also known to exist. Thus, Klafky et al. (1995), Neuroscience Letters 201, 29-32 studied the action of the proteasome inhibitor calpain inhibitor 1 on the secretion of the β-amyloid peptide.
Fenteany et al. (1995), Science, 268, 726-731 describe lactacystin as metabolite of streptomycetes, which acts as a cell cycle inhibitor and leads to the induction of neurite growth in murine neuroblastoma cells. The cellular target for this inhibitor is the 20S proteasome.
Kroll et al. (1999) Chem. Biol. 6, 889-698 describe a fungal metabolite, epipolythiodioxopiperazine (gliotoxin), which suppresses, inter alia, antigen processing in mammalian cells. As the authors were able to show, gliotoxin is a non-competitive inhibitor of the 20S proteasome in vitro.
Mellgren (1997) J. Biol. Chem. 272(47), 29899-29903 describes the inhibitory action of various peptidyl compounds on the human 20S proteasome. This publication further describes that the said substances at concentrations of 200 μM had no effect on the growth of fungal cells (S. cerevisiae). In agreement therewith, Lee and Goldberg (1998), (Proteasome inhibitors cause induction of Heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae, Molecular and Cellular Biology 18, 30-38) demonstrate that pharmacologically highly effective proteasome inhibitors such as MG-132 (Z-Leu-Leu-Leu-CHO) have only a minimum effect, if any, on the growth rate of yeast cells at 30° C.
Stack et al. (2000), Nature Biotechnology 18, 1298-1302 describe a cellular assay system for identifying inhibitors of the proteasome and indicate the possible use of these inhibitors for the treatment of Alzheimer's or Parkinson's disease. Here, the half-life of proteins is shortened by genetically modifying ubiquitin in such a way that proteins labelled therewith are destabilized. If an inhibit prevents protein degradation, it is possible to determine this on the basis of a reporter gene.
WO 00/33654 A1 describes the use of proteasome inhibitors for the treatment of various human disorders. A starting point for the action of the said inhibitors, which is mentioned here, is the immunomodulating activity of the 26S proteasome.
Thus, the proteasome has hitherto been described as a target protein for the treatment of various disorders of the human organism. Whether the proteasome from fungi is also accessible for active compounds and whether it can be inhibited or modulated by the latter, and whether such active compounds may also be used in vivo, i.e. as fungicides, has hitherto neither been studied nor described in detail. Mellgren (1997), J. Biol. Chem. 272(47), 29899-29903 only reveals that the inhibitors of the human proteasome described therein have, at a concentration of 200 μM, no inhibitory action on yeast cells. Lee and Goldberg (1998) furthermore demonstrate that pharmacologically highly effective proteasome inhibitors have only a minimal effect, if any, on the growth rate of yeast cells.
Thus, the prior art only reveals that, although inhibitors of the human proteasome exist, these inhibitors exhibit no action in fungi. The fungal proteasome thus seems inaccessible to the inhibitory action of the compounds. It would nonetheless be desirable to provide new fungicides which have new sites of action and mechanisms in order to prevent thereby, for example, the development of resistance to known fungicides with different sites of action and to develop more effective or more specific, and thereby also environmentally more compatible fungicides.